In order to gain a deeper insight into the genetic regulation of cytokine-determined and immunoglobulin/ T cell receptor based signaling in lymphocytes, efforts to use RNA interference (RNAi) technology as a screening tool have been undertaken. An shRNA library consisting of all the mouse phosphatases has been prepared in lentivirus vectors with a puromycin resistance element. Unit scientists have chosen to analyze the importance of phosphatases in peripheral CD4 T cell differentiation to Th1 cells. The model utilized takes advantage of an indicator mouse prepared in the Unit in which ZS-Green expression marks the presence of the master Th1 transcriptional regulator T-bet. When T cells differentiate in vitro into Th1 cells, they express very large amounts of GFP, are easily detectable and can be purified by cell sorting. The initial screen carried out was aimed at determining what phosphatases were important for the expression or the extinction of T-bet during the Th differentiation process. Nave CD4 T cells were exposed to a library of all shRNAs complementary to phosphatases (over 1000 members), The shRNAs were in lentiviruses so that they could be introduced into naive CD4 T cells. The cells were then differentiated under Th1 conditions for 4 days, rested in IL-2 and exposed to puromycin, to eliminate cells that had not incorporated and expressed a member of the library. At this stage the great majority of the cells were already ZS-Green+. The cells were then shifted to Th2 culture conditions. At the end of four additional days of culture , 1/2 of the former Th1 cells were T-bet negative. The T-bet negative and positive cells were purified and the incorporated shRENAs were PCR amplified, utilizing a method to avoid the difficulties resulting from the hairpin structure. The resulting large set of amplified shRNAs were subjected to deep sequencing using an ABI instrument. More than 20 shRNAs were found to be uniquely expressed in the T-bet negative cells and a similar number in the T-bet positives. The technical aspects were were shown to be reproducible using PCR amplification with one external and one internal primer. MTMR7 and MTMR9, phosphateses that act on phosphatidyl inositols showed reproducible activity both in repetition of the assay used in screening and in priming done under limiting Th1 conditions. Assays indicate that these phosphatases can effect the PI-3 kinase pathway, as shown by their activity to regulate AKT phosphorylation. This work emphasis the key role played by phosphatases in regulating lymphocyte differentiation and the power of library based screening in identifying candiates for further analysis. In parallel, a chemical genomic screen has been undertaken to examine the cpacity of various small molecules from the LOPAC set to alter the rate at which Th17 cells switch to Th1 cells. This has been done using the ZS-Green marker so that small molecules that inhibit or enhance CD4 T cell plasticity have a reasonable likelihood of being detected. Several candidates that inhibit switching have been identified and are now in the course of secondary screening.